Fe(III) reduction by irradiated <i>S</i>. <i>oneidensis</i> MR-1.

<p>Cultures of <i>S</i>. <i>oneidensis</i> (MR-1) were grown aerobically in tryptic soy broth (30°C; 130 rpm) to late log–early stationary phase. Biomass was harvested and washed twice in sterile 30 mM sodium bicarbonate buffer prior to irradiation with 50 Gy X-radiation (rad). Immediately after irradiation, cell suspensions were driven anoxic with an 80:20 gas mix of N2:CO2 prior to inoculation into an anoxic medium containing 20 mM lactate as electron donor, 50 mM Fe(III) as poorly crystalline insoluble Fe(III) oxide and 30 mM sodium bicarbonate. 10 μM riboflavin (Rf) was added to media post-irradiation as an electron shuttle where necessary. Fe(II) concentrations were determined by ferrozine assay after extraction with 0.5 N HCl. Error bars depict standard error of the mean of triplicate experiments.</p

Cluster analysis of spectra from irradiated and control cultures of <i>S</i>. <i>oneidensis</i> MR-1.

<p>Euclidean distances between cluster centres of control and irradiated sample discriminant function scores extracted during principal component-discriminant function analysis (PC-DFA) applied to (A) FT-IR spectra and (B) MALDI mass spectra of lag, exponential and stationary phase X-irradiated and control cultures. Principal components 1 to 5 (FT-IR) and 1 to 30 (MALDI-MS) were used by the DFA algorithm with <i>a priori</i> knowledge of machine replicates, i.e. 1 class per sample point and treatment, giving 6 classes in total for each dose.</p

Schematic diagram of central carbon metabolism in <i>P</i>. <i>putida</i> DOT-T1E-18 adapted to propranolol showing the level of metabolites for cells exposed to propranolol compared to the control ones.

<p>Metabolites were detected and identified by GC-MS. Metabolites indicated in black were observed, while metabolites indicated in grey were not detected. The median <i>m/z</i> intensity (red line) in the box- whisker plots was used to compare the level of metabolites. (A) Represent the level of metabolites at 10 min, while (B) the level of metabolite at 60 min. Traffic light system represents different concentration of propranolol. Red, yellow and green represent exposed cells to 0.2, 0.4 and 0.6 mg/mL of propranolol respectively. Up-arrow, down-arrow and steady arrow indicate an increase, a decrease and no change in the level of metabolite respectively. The number of arrows represents the level of metabolites. Slight change (single arrow), medium change (double arrows) and high change (triple arrows).</p

PC-DFA scores plots of FT-IR data for three different strains of <i>P</i>. <i>putida</i> strains upon propranolol shock.

<p>Symbols represent different strains. (A) <i>P</i>. <i>putida</i> DOT-T1E is the wild type (stars) and ten PCs with a total explained variance (TEV) of 99.43% were used for the DFA, (B) <i>P</i>. <i>putida</i> DOT-T1E-PS28 is the mutant (closed triangles) and ten PCs with a TEV of 99.65% were used for the DFA, (C) <i>P</i>. <i>putida</i> DOT-T1E-18 is the mutant (closed circles) and ten PCs with a TEV of 99.03% were used for the DFA. Colour coding: control with no propranolol (red), cells exposed to 0.2 mg mL^-1 propranolol (black), 0.4 mg mL^-1 propranolol (brown), and 0.6 mg mL^-1 propranolol (blue). Arrows indicate the direction of shift because of the increase of propranolol concentration. (D) PC-DFA loadings plot for <i>P</i>. <i>putida</i> DOT-T1E. (E) PC-DFA loadings plot for <i>P</i>. <i>putida</i> DOT-T1E-PS28, (F) PC-DFA loadings plot for <i>P</i>. <i>putida</i> DOT-T1E-18. Significant loadings were assigned to bacterial proteins.</p

Growth, survival and extension of lag phase in X-irradiated cultures of <i>S</i>. <i>oneidensis</i> MR-1.

<p>(A) Growth profiles of aerobic cultures of <i>S</i>. <i>oneidensis</i> MR-1 (30°C) after exposure to 12, 24, 48, 72 and 95 Gy X-radiation (0.79 Gy min^-1). Irradiations began at t = 0. A minimal growth medium was used, based on that described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131249#pone.0131249.ref032" target="_blank">32</a>]. Data points show mean of triplicate batch cultures and error bars depict 95% confidence intervals. (B) Mean time difference in lag phase duration between irradiated cultures and respective controls (measured at mid exponential phase). Error bars depict 95% confidence intervals from three biological replicates. (C) Survival of <i>S</i>. <i>oneidensis</i> MR-1 exposed to acute doses of X-radiation. Cultures were irradiated in the growth medium described above, serially diluted in phosphate buffered saline and plated on to solid growth medium (same as above with 1.5% agar). Error bars depict standard error of the mean CFU mL^-1.</p

Box-whisker plot showing the changes in ornithine levels (variable id 100) in control and exposed cells to propranolol.

<p>(Red line) indicates the median <i>m/z</i> intensity. These plots represent the data for 3 <i>P</i>. <i>putida</i> strains, 4 concentrations of propranolol and 3 time points, for 4 biological replicates. Dashed lines separate different concentration levels of propranolol and solid line separates different strains.</p

Schematic metabolic diagram of central carbon metabolism in <i>P</i>. <i>putida</i> DOT-T1E adapted to propranolol showing the level of metabolites for cells exposed to propranolol compared to the control.

<p>Metabolites were detected and identified by GC-MS. Metabolites indicated in black were observed, while metabolites indicated in grey were not detected. The median <i>m/z</i> intensity (red line) in the box- whisker plots was used to compare the level of metabolites. (A) Represent the level of metabolites at 10 min, while (B) the level of metabolite at 60 min. Traffic light system represents different concentration of propranolol. Red, yellow and green represent exposed cells to 0.2, 0.4 and 0.6 mg/mL of propranolol respectively. Up-arrow, down-arrow and steady arrow indicate an increase, a decrease and no change in the level of metabolite respectively. The number of arrows represents the level of metabolites. Slight change (single arrow), medium change (double arrows) and high change (triple arrows).</p

Radiation induced changes to proteins in cultures of X-irradiated <i>S</i>. <i>oneidensis</i> MR-1.

<p>Mean MALDI-MS spectra of <i>S</i>. <i>oneidensis</i> MR-1 exposed to (A) 12 Gy X-radiation and (B) 95 Gy X-radiation. Spectra have been offset in the <i>y</i>-axis so that spectral features are clearly visible. Asterisks (*) show mass peaks in irradiated spectra that are discriminant with respect to batch controls, as observed in difference spectra (mean irradiated spectrum minus mean control spectrum) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131249#pone.0131249.s007" target="_blank">S7 Fig</a>). The mass range has been limited in the figure to only include peaks which are discriminant and to which tentative annotations can be assigned, displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131249#pone.0131249.t001" target="_blank">Table 1</a>.</p

Partial least squares regression analysis of FT-IR spectra from control and irradiated cultures of <i>S</i>. <i>oneidensis</i> MR-1.

<p>Scores for the first two principal components (PC1 and PC2) extracted during partial least squares regression analysis performed on FT-IR data of control and X-irradiated cultures at lag phase. Solid black circles represent control samples and crosses represent irradiated samples. The nine replicates from each treatment are formed from three experimental replicates from each biological replicate.</p

Box-whisker plots showing the changes in metabolite levels in control and cells exposed to propranolol for 4 <i>biological</i> replicates.

<p>Variable 180 was identified as propranolol. (Red line) indicates the median <i>m/z</i> intensity. (A) Represent the data for 3 <i>P</i>. <i>putida</i> strains, 4 concentrations of propranolol and 3 time points, dashed lines separate different concentration levels of propranolol and solid line separates different strains. (B) Represent the data for 3 <i>P</i>. <i>putida</i> strains, 3 concentrations of propranolol and 1 time point at 60 min, dashed lines separates different strains.</p